Crystal forms of kinase inhibitors

ABSTRACT

Citrate salts of N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea and crystalline forms thereof are suitable pharmaceutical ingredients for pharmaceutical compositions useful in the treatment of disease, for example, cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/353,038 filed Jun. 9, 2010, which is incorporated by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to crystalline forms of citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,processes for preparing the crystalline forms, pharmaceuticalformulations thereof, and methods of treating cancer.

BACKGROUND OF THE INVENTION

Mitosis is a process by which a complete copy of a duplicated genome issegregated by the microtuble spindle apparatus into two daughter cells.Aurora-kinases, key mitotic regulators required for genome stability,have been found to be overexpressed in human tumors. There is thereforean existing need in the therapeutic arts for compounds which inhibitAurora-kinases, compositions comprising the inhibitors and methods oftreating diseases during which Aurora-kinases are unregulated oroverexpressed.

The reversible phosphorylation of proteins is one of the primarybiochemical mechanisms mediating eukaryotic cell signaling. Thisreaction is catalyzed by protein kinases that transfer the g-phosphategroup of ATP to hydroxyl groups on target proteins. 518 such enzymesexist in the human genome of which ˜90 selectively catalyze thephosphorylation of tyrosine hydroxyl groups. Cytosolic tyrosine kinasesreside intracellularly whereas receptor tyrosine kinases (RTKs) possessboth extracellular and intracellular domains and function as membranespanning cell surface receptors. As such, RTKs mediate the cellularresponses to environmental signals and facilitate a broad range ofcellular processes including proliferation, migration and survival.

RTK signaling pathways are normally highly regulated, yet theirover-activation has been shown to promote the growth, survival andmetastasis of cancer cells. Dysregulated RTK signaling occurs throughgene over-expression or mutation and has been correlated with theprogression of various human cancers.

The VEGF receptor (VEGFR) family consists of three RTKs, KDR (kinaseinsert domain-containing receptor; VEGFR2), FLT1 (Fms-like tyrosinekinase; VEGFR1), and FLT4 (VEGFR3). These receptors mediate thebiological function of the vascular endothelial growth factors (VEGF-A,-B, -C, -D, -E and placenta growth factor (PlGF)), a family ofhomodimeric glycoproteins that bind the VEGF receptors with varyingaffinities.

KDR is the major mediator of the mitogenic, angiogenic andpermeability-enhancing effects of VEGF-A, hereafter referred to as VEGF.Many different cell types are able to produce VEGF, yet its biologicalactivity is limited predominately to the vasculature by way of theendothelial cell-selective expression of KDR. Not surprisingly, theVEGF/KDR axis is a primary mediator of angiogenesis, the means by whichnew blood vessels are formed from preexisting vessels.

FLT1 binds VEGF, VEGF-B and placental growth factor. FLT1 is expressedon the surface of smooth muscle cells, monocytes and hematopoietic stemscells in addition to endothelial cells. Activation of FLT1 signalingresults in the mobilization of marrow-derived endothelial progenitorcells that are recruited to tumors where they contribute to new bloodvessel formation.

FLT4 mediates the signaling of VEGF-C and VEGF-D, which mediateformation of tumor-associated lymphatic vessels (lymphangiogenesis).Lymphatic vessels are one of the routes by which cancer cellsdisseminate from solid tumors during metastasis.

The PDGF receptor (PDGFR) family consists of five RTK's, PDGFR-a and -b,CSF1R, KIT, and FLT3.

CSF-1R is encoded by the cellular homolog of the retroviral oncogenev-fms and is a major regulator of macrophage development. Macrophagesare frequent components of tumor stroma and have been shown to modifythe extracellular matrix in a manner beneficial to tumor growth andmetastasis.

KIT is expressed by hematopoietic progenitor cells, mast cells, germcells and by pacemaker cells in the gut (interstitial cells of Cajal).It contributes to tumor progression by two general mechanisms namelyautocrine stimulation by its ligand, stem cell factor (SCF), and throughmutations that result in ligand-independent kinase activity.

FLT3 is normally expressed on hematopoietic stem cells where itsinteraction with FLT3 ligand (FL) stimulates stem cell survival,proliferation and differentiation. In addition to being over-expressedin various leukemia cells, FLT3 is frequently mutated in hematologicalmalignancies with approximately one-third of patients with acute myeloidleukemia (AML) harboring activating mutations.

The identification of effective small compounds which specificallyinhibit signal transduction and cellular proliferation by modulating theactivity of tyrosine kinases to regulate and modulate abnormal orinappropriate cell proliferation, differentiation, or metabolism istherefore desirable. In particular, the identification of methods andcompounds that specifically inhibit the function of a tyrosine kinasewhich is essential for angiogenic processes or the formation of vascularhyperpermeability leading to edema, ascites, effusions, exudates, andmacromolecular extravasation and matrix deposition as well as associateddisorders would be beneficial.

Compounds that inhibit protein kinases such as Aurora-kinases and theVEGFR and PDGFR families of kinases have been identified, includingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea.These compounds, and methods to make them, are disclosed in U.S. patentapplication Ser. No. 12/632,183 (hereinafter “the '183 application”),incorporated by reference herein in its entirety.

It now has been found that citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureacan be converted into crystalline forms, which can advantageously beused as an active pharmaceutical ingredient in cancer therapy. To thatpurpose, these crystalline forms are converted into pharmaceuticalformulations.

A crystalline form is the form in which the position of the moleculesrelative to one another is organized according to a three-dimensionallattice structure. Polymorphs are different crystalline forms of thesame compound resulting from a different arrangement of the molecules inthe solid state. Polymorphs differ from each other in their physicalproperties but not their chemical composition.

Polymorphism is of particular interest in the development of suitablepharmaceutical dosage forms. Certain polymorphic forms may exhibitsuperior stability and storability, resulting in enhanced shelf-life ofthe pharmaceutical product. In addition, certain polymorphic forms aremore readily manufactured in high purity in large quantities.

Critically, polymorphs of an active pharmaceutical ingredient can havedifferent aqueous solubility and dissolution rates, which may havetherapeutic consequences due to the potential differences inbioavailability between polymorphs of the same compound.

The present invention provides crystalline forms of citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahaving beneficial properties in one or more of the followingcharacteristics: the ability to be formulated in a pharmaceutical dosageform, adequate shelf-life in a pharmaceutical dosage form, and/orability to be effectively administered in a pharmaceutical dosage form.

SUMMARY OF THE INVENTION

In one embodiment, the invention providesN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate in solid crystalline form.

In one embodiment, the invention providesN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in solid crystalline form.

In a further embodiment, the invention provides a crystal polymorph ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate characterized herein and designated dihydrogencitrate Form I.

In a further embodiment, the invention provides hydrated forms ofdihydrogen citrate Form I, including the monohydrate form.

In a further embodiment, the invention provides a crystal polymorph ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate characterized herein and designated hydrogen citrateForm II.

In a further embodiment, the invention provides hydrated forms ofhydrogen citrate Form II, including the monohydrate form.

There is further provided a pharmaceutical composition comprisingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I and one or more pharmaceutically acceptableexcipients.

There is further provided a pharmaceutical composition comprisingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II and one or more pharmaceutically acceptableexcipients.

There is further provided process for preparingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I comprising providing a mixture comprisingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,water, acetone, and citric acid, and causing theN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I to exist in the mixture.

There is further provided process for preparingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II comprising providing a mixture comprisingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,water, tetrahydrofuran, and citric acid, and causing theN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II to exist in the mixture.

In a further embodiment, the invention provides a method for treatingcancer in a mammal comprising administering to a subject having thedisease therapeutically effective amount of citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureain a solid crystalline form, wherein the crystalline form is Form I orII, or (b) a pharmaceutical composition comprising citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureain a solid crystalline form, wherein the crystalline form is Form I orII, one or more pharmaceutically acceptable excipients. Examples of suchcancers include myelodysplastic syndrome, acute myeloid leukemia,colorectal cancer, non-small cell lung cancer, and ovarian cancer.

There is still further provided a method for treating cancer in a mammalcomprising dissolvingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate in a solid crystalline form, wherein the crystallineform is Form I, in a pharmaceutically acceptable solvent or mixture ofsolvents, and administering the resulting solution in a therapeuticallyeffective amount to subject having the disease.

There is still further provided a method for treating cancer in a mammalcomprising dissolvingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate in a solid crystalline form, wherein the crystallineform is Form I, in a pharmaceutically acceptable solvent or mixture ofsolvents, and administering the resulting solution in a therapeuticallyeffective amount to subject having the disease.

There is still further provided a method for treating cancer in a mammalcomprising dispersingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in a solid crystalline form, wherein the crystallineform is Form II, in a pharmaceutically acceptable polymeric carrier, andadministering the resulting solid dispersion in a therapeuticallyeffective amount to subject having the disease.

There is still further provided a method for treating cancer in a mammalcomprising dissolvingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in a solid crystalline form, wherein the crystallineform is Form II, in a pharmaceutically acceptable solvent or mixture ofsolvents, and administering the resulting solution in a therapeuticallyeffective amount to subject having the disease.

Additional embodiments of the invention, including more particularaspects of those provided above, will be found in, or will be evidentfrom, the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a PXRD scan of crystal polymorph Form IN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate.

FIG. 2 is a PXRD scan of crystal polymorph Form IIN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate.

DETAILED DESCRIPTION

The invention encompasses crystalline forms of citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea.N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureafree base is prepared, illustratively, as described in Example 1 ofabove-cited U.S. patent application Ser. No. 12/632,183, the entiredisclosure of which is incorporated by reference herein. The term “freebase” is used for convenience herein to refer toN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureaparent compound as distinct from any salt thereof.

Those skilled in the art will also understand that the term“monohydrate” when referring toN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureameans that there is one water molecule for every molecule ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea.

In one embodiment, the invention encompassesN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate in a solid crystalline form.

In one embodiment the invention encompasses a crystalline form ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate, characterized at least by a powder X-ray diffractionpeak at any one or more of the following positions: 11.80, 14.59, 15.95,21.38, 26.12° 2θ, ±0.2° 2θ, herein defined as Form I.

Another aspect of the invention encompasses a crystalline form ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I, characterized at least by a powder X-raydiffraction peak at each of the following positions: 11.80, 14.59,15.95, 21.38, 26.12° 2θ, ±0.2° 2θ.

Another aspect of the invention encompasses a crystalline form ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I, characterized at least by a powder X-raydiffraction peak at each of the following positions: 7.56, 7.96, 11.80,12.42, 13.44, 14.59, 15.66, 15.95, 16.69, 21.38, 22.40, 22.82, 23.98,24.50, 24.99, 26.12° 2θ, ±0.2° 2θ.

In another embodiment, the invention encompasses Form I in the form ofthe monohydrate.

In another embodiment, the invention provides a process for preparingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate in a solid crystalline form, wherein the crystallineform is Form I. The process comprises a) providing a mixture comprising(i)N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,water, acetone, and citric acid; b) causingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate crystalline Form I to exist in the mixture. Theprocess can further comprise isolatingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate in a solid crystalline form, wherein the crystallineform is Form I.

The invention further comprises a pharmaceutical composition comprisingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I and one or more pharmaceutically acceptableexcipients.

In one embodiment, the invention encompassesN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in a solid crystalline form.

In another embodiment, the invention encompasses the compoundN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in a solid crystalline form, wherein the crystallineform is Form II, characterized at least by a powder X-ray diffractionpeak at any one or more of the following positions: 8.65, 15.18, 24.47,28.02° 2θ, ±0.2° 2θ.

Another aspect of the invention encompasses a crystalline form ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II, characterized at least by a powder X-raydiffraction peak at each of the following positions: 8.65, 15.18, 24.47,28.02° 2θ, ±0.2° 2θ.

Another aspect of the invention encompasses a crystalline form ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II, characterized at least by a powder X-raydiffraction peak at each of the following positions: 5.71, 6.30, 7.27,8.65, 9.80, 13.06, 14.31, 15.18, 15.92, 16.68, 17.71, 20.17, 21.98,23.30, 24.47, 26.11, 28.02° 2θ, ±0.2° 2θ.

In another embodiment, the invention provides a process for preparingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in a solid crystalline form, wherein the crystallineform is Form II. The process comprises a) providing a mixture comprising(i)N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,water, tetrahydrofuran, and citric acid; and b) causingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate crystalline Form I to exist in the mixture. The processcan further comprise isolatingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate in a solid crystalline form, wherein the crystallineform is Form II.

The invention further comprises a pharmaceutical composition comprisingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II and one or more pharmaceutically acceptableexcipients.

The crystalline form of citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,for example Form I or II, can be useful as API for the preparation ofpharmaceutical compositions suitable for any route of administration,including oral, to a subject in need thereof. Other routes ofadministration include, without limitation, parenteral, sublingual,buccal, intranasal, pulmonary, topical, transdermal, intradermal,ocular, otic, rectal, vaginal, intragastric, intracranial, intrasynovialand intra-articular routes.

Where it is desired to provide citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea,for example Form I or II, in solution form, for example in a liquidformulation for oral or parenteral administration, the citrate salts ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureawill not, of course, be present in such a formulation in crystallineform; indeed, the presence of crystals is generally undesired in such aformulation. However, crystallineN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate or hydrogen citrate of the present invention cannonetheless be important as API in a process for preparing such aformulation.

Even where the desired formulation is one containingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate or hydrogen citrate in amorphous form, for example asolid dispersion formulation, crystallineN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate or hydrogen citrate can still be useful as API in aprocess for preparing such a formulation.

As API, a crystalline form ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate or hydrogen citrate such as Form I or II hasadvantages over the amorphous form. For example, purification of API tothe high degree of purity required by most regulatory authorities ismore efficient and therefore costs less where the API is crystalline asopposed to amorphous form. Physical and chemical stability, andtherefore shelf-life of the API solid, is also typically better forcrystalline than amorphous forms. Ease of handling is improved over theamorphous form, which tends to be oily or sticky. Drying is morestraightforward and more easily controlled in the case of thecrystalline material, which has a well-defined drying or desolvationtemperature, than in the case of the amorphous material, which hasgreater affinity for organic solvents and no well-defined dryingtemperature. Downstream processing using crystalline API permitsenhanced process control. These advantages are illustrative anon-limiting.

N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureais present in a pharmaceutical composition of the invention in an amountthat can be therapeutically effective when the composition isadministered to a subject in need thereof according to an appropriateregimen. Typically, a unit dose (the amount administered at a singletime), which can be administered at an appropriate frequency, e.g.,twice daily to once weekly, is about 10 to about 1,000 mg, depending onthe compound in question. Where frequency of administration is oncedaily (q.d.), unit dose and daily dose are the same. Illustratively, theunit dose is typically about 25 to about 1,000 mg, more typically about50 to about 500 mg, for example about 50, about 100, about 150, about200, about 250, about 300, about 350, about 400, about 450 or about 500mg.

Excipients include but are not limited to, for example, encapsulatingmaterials and additives such as absorption accelerators, antioxidants,binders, buffers, carriers, coating agents, coloring agents, diluents,disintegrating agents, emulsifiers, extenders, fillers, flavoringagents, glidants, humectants, lubricants, perfumes, preservatives,propellants, releasing agents, sterilizing agents, sweeteners,solubilizers, wetting agents, mixtures thereof and the like.

Excipients for preparation of formulations comprising or made withcrystallineN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II to be administeredorally in solid dosage form include, for example, agar, alginic acid,aluminum hydroxide, benzyl alcohol, benzyl benzoate, 1,3-butyleneglycol, carbomers, castor oil, cellulose, cellulose acetate, cocoabutter, copovidone, corn starch, corn oil, cottonseed oil,cross-povidone, diglycerides, ethanol, ethyl cellulose, ethyl laureate,ethyl oleate, fatty acid esters, gelatin, germ oil, glucose, glycerol,groundnut oil, hydroxypropylmethyl cellulose, isopropanol, isotonicsaline, lactose, magnesium hydroxide, magnesium stearate, malt,mannitol, monoglycerides, olive oil, povidone, peanut oil, potassiumphosphate salts, potato starch, povidone, propylene glycol, Ringer'ssolution, safflower oil, sesame oil, silicon dioxide, sodiumcarboxymethyl cellulose, sodium phosphate salts, sodium lauryl sulfate,sodium sorbitol, sodium stearylfumarate, soybean oil, stearic acids,stearyl fumarate, sucrose, surfactants, talc, tragacanth,tetrahydrofurfuryl alcohol, triglycerides, vitamin E and derivativesthereof, water, mixtures thereof and the like.

Excipients for preparation of compositions comprising or made withN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II to be administeredophthalmically or orally in liquid dosage forms include, for example,1,3-butylene glycol, castor oil, corn oil, cottonseed oil, ethanol,fatty acid esters of sorbitan, germ oil, groundnut oil, glycerol,isopropanol, olive oil, polyethylene glycols, propylene glycol, sesameoil, water, mixtures thereof and the like.

Excipients for preparation of compositions comprising or made withN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II to be administeredosmotically include, for example, chlorofluorohydrocarbons, ethanol,water, mixtures thereof and the like.

Excipients for preparation of compositions comprising or made withN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II to be administeredparenterally include, for example, 1,3-butanediol, castor oil, corn oil,cottonseed oil, dextrose, germ oil, groundnut oil, liposomes, oleicacid, olive oil, peanut oil, Ringer's solution, safflower oil, sesameoil, soybean oil, U.S.P. or isotonic sodium chloride solution, water,mixtures thereof and the like.

Excipients for preparation of compositions comprising or made withN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II to be administeredrectally or vaginally include, but are not limited to, cocoa butter,polyethylene glycol, wax, mixtures thereof and the like.

The composition is normally administered in an amount providing atherapeutically effective daily dose of the drug. The term “daily dose”herein means the amount of drug administered per day, regardless of thefrequency of administration. For example, if the subject receives a unitdose of 150 mg twice daily, the daily dose is 300 mg. Use of the term“daily dose” will be understood not to imply that the specified dosageamount is necessarily administered once daily. However, in a particularembodiment the dosing frequency is once daily (q.d.), and the daily doseand unit dose are in this embodiment the same thing.

What constitutes a therapeutically effective dose depends on theparticular compound, the subject (including species and body weight ofthe subject), the disease (e.g., the particular type of cancer) to betreated, the stage and/or severity of the disease, the individualsubject's tolerance of the compound, whether the compound isadministered in monotherapy or in combination with one or more otherdrugs, e.g., other chemotherapeutics for treatment of cancer, and otherfactors. Thus the daily dose can vary within wide margins, for examplefrom about 10 to about 1,000 mg. Greater or lesser daily doses can beappropriate in specific situations. It will be understood thatrecitation herein of a “therapeutically effective” dose herein does notnecessarily require that the drug be therapeutically effective if only asingle such dose is administered; typically therapeutic efficacy dependson the composition being administered repeatedly according to a regimeninvolving appropriate frequency and duration of administration. It isstrongly preferred that, while the daily dose selected is sufficient toprovide benefit in terms of treating the cancer, it should not besufficient to provoke an adverse side-effect to an unacceptable orintolerable degree. A suitable therapeutically effective dose can beselected by the physician of ordinary skill without undueexperimentation based on the disclosure herein and on art cited herein,taking into account factors such as those mentioned above. The physicianmay, for example, start a cancer patient on a course of therapy with arelatively low daily dose and titrate the dose upwards over a period ofdays or weeks, to reduce risk of adverse side-effects.

Illustratively, suitable doses ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureaare generally about 10 to about 1,000 mg/day, more typically about 50 toabout 500 mg/day or about 200 to about 400 mg/day, for example about 50,about 100, about 150, about 200, about 250, about 300, about 350, about400, about 450 or about 500 mg/day, administered at an average dosageinterval of 3 to 10 days, or about 4 to 8 days, or about 7 days.

A composition comprising crystallineN-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II (or prepared usingas API) of the invention are suitable for use in monotherapy or incombination therapy, for example with other chemotherapeutics or withionizing radiation.

A composition comprising crystallineN-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II (or prepared usingas API), can be administered in combination therapy with one or moretherapeutic agents that include, but are not limited to, alkylatingagents, angiogenesis inhibitors, antibodies, antimetabolites,antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors,other apoptosis promoters (for example, Bcl-xL, Bcl-w and Bfl-1inhibitors), activators of a death receptor pathway, Bcr-Abl kinaseinhibitors, BiTE (bi-specific T-cell engager) antibodies, antibody-drugconjugates, biological response modifiers, cyclin-dependent kinase (CDK)inhibitors, cell cycle inhibitors, cyclooxygenase-2 (COX-2) inhibitors,dual variable domain binding proteins (DVDs), human epidermal growthfactor receptor 2 (ErbB2 or HER/2neu) receptor inhibitors, growth factorinhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase(HDAC) inhibitors, hormonal therapies, immunologicals, inhibitors ofapoptosis proteins (IAPB), intercalating antibiotics, kinase inhibitors,kinesin inhibitors, JAK2 inhibitors, mammalian target of rapamycin(mTOR) inhibitors, microRNAs, mitogen-activated extracellularsignal-regulated kinase (MEK) inhibitors, multivalent binding proteins,non-steroidal anti-inflammatory drugs (NSAIDs), poly-ADP (adenosinediphosphate)-ribose polymerase (PARP) inhibitors, platinumchemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs,pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids,deltoids, plant alkaloids, small inhibitory ribonucleic acids (siRNAs),topoisomerase inhibitors, ubiquitin ligase inhibitors, and the like.

BiTE antibodies are bi-specific antibodies that direct T-cells to attackcancer cells by simultaneously binding the two cells. The T-cell thenattacks the target cancer cell. Examples of BiTE antibodies include, butare not limited to, adecatumumab (Micromet MT201), blinatumomab(Micromet MT103) and the like. Without being limited by theory, one ofthe mechanisms by which T-cells elicit apoptosis of the target cancercell is by exocytosis of cytolytic granule components, which includeperform and granzyme B. In this regard, Bcl-2 has been shown toattenuate the induction of apoptosis by both perform and granzyme B.These data suggest that inhibition of Bcl-2 could enhance the cytotoxiceffects elicited by T-cells when targeted to cancer cells (Sutton et al.(1997) J. Immunol. 158:5783-5790).

SiRNAs are molecules having endogenous RNA bases or chemically modifiednucleotides. The modifications do not abolish cellular activity, butrather impart increased stability and/or increased cellular potency.Examples of chemical modifications include phosphorothioate groups,2′-deoxynucleotide, 2′-OCH₃-containing ribonucleotides,2′-F-ribonucleotides, 2′-methoxyethyl ribonucleotides, combinationsthereof and the like. The siRNA can have varying lengths (e.g., 10-200bps) and structures (e.g., hairpins, single/double strands, bulges,nicks/gaps, mismatches) and are processed in cells to provide activegene silencing. A double-stranded siRNA (dsRNA) can have the same numberof nucleotides on each strand (blunt ends) or asymmetric ends(overhangs). The overhang of 1-2 nucleotides can be present on the senseand/or the antisense strand, as well as present on the 5′- and/ or the3′-ends of a given strand. For example, siRNAs targeting Mcl-1 have beenshown to enhance the activity of ABT-263 or ABT-737 in various tumorcell lines (Tse et al. (2008) Cancer Res. 68:3421-3428 and referencestherein).

Multivalent binding proteins are binding proteins comprising two or moreantigen binding sites. Multivalent binding proteins are engineered tohave the three or more antigen binding sites and are generally notnaturally occurring antibodies. The term “multispecific binding protein”means a binding protein capable of binding two or more related orunrelated targets. Dual variable domain (DVD) binding proteins aretetravalent or multivalent binding proteins binding proteins comprisingtwo or more antigen binding sites. Such DVDs may be monospecific (i.e.,capable of binding one antigen) or multispecific (i.e., capable ofbinding two or more antigens). DVD binding proteins comprising twoheavy-chain DVD polypeptides and two light-chain DVD polypeptides arereferred to as DVD Ig's. Each half of a DVD Ig comprises a heavy-chainDVD polypeptide, a light-chain DVD polypeptide, and two antigen bindingsites. Each binding site comprises a heavy-chain variable domain and alight-chain variable domain with a total of 6 CDRs involved in antigenbinding per antigen binding site.

Alkylating agents include altretamine, AMD-473, AP-5280, apaziquone,bendamustine, brostallicin, busulfan, carboquone, carmustine (BCNU),chlorambucil, Cloretazine™ (laromustine, VNP 40101M), cyclophosphamide,dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide,KW-2170, lomustine (CCNU), mafosfamide, melphalan, mitobronitol,mitolactol, nimustine, nitrogen mustard N-oxide, ranimustine,temozolomide, thiotepa, treosulfan, trofosfamide and the like.

Angiogenesis inhibitors include epidermal growth factor receptor (EGFR)inhibitors, endothelial-specific receptor tyrosine kinase (Tie-2)inhibitors, insulin growth factor-2 receptor (IGFR-2) inhibitors, matrixmetalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9(MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR)inhibitors, thrombospondin analogs, vascular endothelial growth factorreceptor tyrosine kinase (VEGFR) inhibitors and the like.

Antimetabolites include Alimta™ (pemetrexed disodium, LY231514, MTA),5-azacitidine, Xeloda™ (capecitabine), carmofur, Leustat™ (cladribine),clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside,decitabine, deferoxamine, doxifluridine, eflornithine, EICAR(5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide), enocitabine,ethenylcytidine, fludarabine, 5-fluorouracil (5-FU) alone or incombination with leucovorin, Gemzar™ (gemcitabine), hydroxyurea,Alkeran™ (melphalan), mercaptopurine, 6-mercaptopurine riboside,methotrexate, mycophenolic acid, nelarabine, nolatrexed, ocfosfate,pelitrexol, pentostatin, raltitrexed, ribavirin, S-1, triapine,trimetrexate, TS-1, tiazofurin, tegafur, vidarabine, UFT and the like.

Antivirals include ritonavir, hydroxychloroquine and the like.

Aurora kinase inhibitors include ABT-348, AZD-1152, MLN-8054, VX-680,aurora A-specific kinase inhibitors, aurora B-specific kinaseinhibitors, pan-aurora kinase inhibitors and the like.

Bcl-2 family protein inhibitors other than ABT-263 or compounds ofFormula I herein include AT-101 ((−)gossypol), Genasense™Bcl-2-targeting antisense oligonucleotide (G3139 or oblimersen),IPI-194, IPI-565,N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide)(ABT-737), GX-070 (obatoclax) and the like.

Bcr-Abl kinase inhibitors include dasatinib (BMS-354825), Gleevec™(imatinib) and the like.

CDK inhibitors include AZD-5438, BMI-1040, BMS-387032, CVT-2584,flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib(CYC-202 or R-roscovitine), ZK-304709 and the like.

COX-2 inhibitors include ABT-963, Arcoxia™ (etoricoxib), Bextra™(valdecoxib), BMS-347070, Celebrex™ (celecoxib), COX-189 (lumiracoxib),CT-3, Deramaxx™ (deracoxib), JTE-522,4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl)-1H-pyrrole, MK-663(etoricoxib), NS-398, parecoxib, RS-57067, SC-58125, SD-8381, SVT-2016,S-2474, T-614, Vioxx™ (rofecoxib) and the like.

EGFR inhibitors include ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine,EMD-7200, Erbitux™ (cetuximab), HR3, IgA antibodies, Iressa™(gefitinib), Tarceva™ (erlotinib or OSI-774), TP-38, EGFR fusionprotein, Tykerb™ (lapatinib) and the like.

ErbB2 receptor inhibitors include CP-724714, CI-1033 (canertinib),Herceptin™ (trastuzumab), Tykerb™ (lapatinib), Omnitarg™ (2C4,petuzumab), TAK-165, GW-572016 (ionafamib), GW-282974, EKB-569, PI-166,dHER2 (HER2 vaccine), APC-8024 (HER2 vaccine), anti-HER/2neu bispecificantibody, B7.her2IgG3, AS HER2 trifunctional bispecific antibodies, mABAR-209, mAB 2B-1 and the like.

Histone deacetylase inhibitors include depsipeptide, LAQ-824, MS-275,trapoxin, suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid andthe like.

HSP-90 inhibitors include 17AAG, CNF-101, CNF-1010, CNF-2024, 17-DMAG,geldanamycin, IPI-504, KOS-953, Mycograb™ (human recombinant antibody toHSP-90), nab-17AAG, NCS-683664, PU24FCl, PU-3, radicicol, SNX-2112,STA-9090, VER-49009 and the like.

Inhibitors of apoptosis proteins include HGS-1029, GDC-0145, GDC-0152,LCL-161, LBW-242 and the like.

Antibody-drug conjugates include anti-CD22-MC-MMAF, anti-CD22-MC-MMAE,anti-CD22-MCC-DM1, CR-011-vcMMAE, PSMA-ADC, MEDI-547, SGN-19A, SGN-35,SGN-75 and the like.

Activators of death receptor pathway include TRAIL and antibodies orother agents that target TRAIL or death receptors (e.g., DR4 and DR5)such as apomab, conatumumab, ETR2-ST01, GDC0145 (lexatumumab), HGS-1029,LBY-135, PRO-1762, trastuzumab and the like.

Kinesin inhibitors include Eg5 inhibitors such as AZD-4877 and ARRY-520,CENPE inhibitors such as GSK-923295A, and the like.

JAK2 inhibitors include CEP-701 (lesaurtinib), XL019, INCB-018424 andthe like.

MEK inhibitors include ARRY-142886, ARRY-438162, PD-325901, PD-98059 andthe like.

mTOR inhibitors include AP-23573, CCI-779, everolimus, RAD-001,rapamycin, temsirolimus, ATP-competitive TORC1/TORC2 inhibitors,including PI-103, PP242, PP30 and Torin 1, and the like.

Non-steroidal anti-inflammatory drugs include Amigesic™ (salsalate),Dolobid™ (diflunisal), Motrin™ (ibuprofen), Orudis™ (ketoprofen),Relafen™ (nabumetone), Feldene™ (piroxicam), ibuprofen cream, Aleve™ andNaprosyn™ (naproxen), Voltaren™ (diclofenac), Indocin™ (indomethacin),Clinoril™ (sulindac), Tolectin™ (tolmetin), Lodine™ (etodolac), Toradol™(ketorolac), Daypro™ (oxaprozin) and the like.

PDGFR inhibitors include CP-673451, CP-868596 and the like.

Platinum chemotherapeutics include cisplatin, Eloxatin™ (oxaliplatin),eptaplatin, lobaplatin, nedaplatin, Paraplatin™ (carboplatin),picoplatin, satraplatin and the like.

Polo-like kinase inhibitors include BI-2536 and the like.

Phosphoinositide-3 kinase inhibitors include wortmannin, LY-294002,XL-147, CAL-120, ONC-21, AEZS-127, ETP-45658, PX-866, GDC-0941, BGT226,BEZ235, XL765 and the like.

Thrombospondin analogs include ABT-510, ABT-567, ABT-898, TSP-1 and thelike.

VEGFR inhibitors include Avastin™ (bevacizumab), ABT-869, AEE-788,Angiozyme™ (a ribozyme that inhibits angiogenesis (RibozymePharmaceuticals (Boulder, Colo.) and Chiron (Emeryville, Calif.)),axitinib (AG-13736), AZD-2171, CP-547632, IM-862, Macugen™ (pegaptanib),Nexavar™ (sorafenib, BAY43-9006), pazopanib (GW-786034), vatalanib(PTK-787 or ZK-222584), Sutent™ (sunitinib or SU-11248), VEGF trap,Zactima™ (vandetanib or ZD-6474) and the like.

Antibiotics include intercalating antibiotics such as aclarubicin,actinomycin D, amrubicin, annamycin, Adriamycin™ (doxorubicin),Blenoxane™ (bleomycin), daunorubicin, Caelyx™ and Myocet™ (liposomaldoxorubicin), elsamitrucin, epirubicin, glarubicin, idarubicin,mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin,rebeccamycin, stimalamer, streptozocin, Valstar™ (valrubicin),zinostatin and the like.

Topoisomerase inhibitors include aclarubicin, 9-aminocamptothecin,amonafide, amsacrine, becatecarin, belotecan, BN-80915, Camptosar™(irinotecan hydrochloride), camptothecin, Cardioxane™ (dexrazoxane),diflomotecan, edotecarin, Ellence™ and Pharmorubicin™ (epirubicin),etoposide, exatecan, 10-hydroxycamptothecin, gimatecan, lurtotecan,mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan, sobuzoxane,SN-38, tafluposide, topotecan and the like.

Antibodies include Avastin™ (bevacizumab), CD40-specific antibodies,chTNT-1/B, denosumab, Erbitux™ (cetuximab), Humax-CD4™ (zanolimumab),IGF1R-specific antibodies, lintuzumab, Panorex™ (edrecolomab), Rencarex™(WX G250), Rituxan™ (rituximab), ticilimumab, trastuzumab, CD20antibodies types I and II and the like.

Hormonal therapies include Arimidex™ (anastrozole), Aromasin™(exemestane), arzoxifene, Casodex™ (bicalutamide), Cetrotide™(cetrorelix), degarelix, deslorelin, Desopan™ (trilostane),dexamethasone, Drogenil™ (flutamide), Evista™ (raloxifene), Afema™(fadrozole), Fareston™ (toremifene), Faslodex™ (fulvestrant), Femara™(letrozole), formestane, glucocorticoids, Hectorol™ (doxercalciferol),Renagel™ (sevelamer carbonate), lasofoxifene, leuprolide acetate,Megace™ (megestrol), Mifeprex™ (mifepristone), Nilandron™ (nilutamide),tamoxifen including Nolvadex™ (tamoxifen citrate), Plenaxis™ (abarelix),prednisone, Propecia™ (finasteride), rilostane, Suprefact™ (buserelin),luteinizing hormone releasing hormone (LHRH) including Trelstar™(triptorelin), histrelin including Vantas™ (histrelin implant),Modrastane™ (trilostane), Zoladex™ (goserelin) and the like.

Deltoids and retinoids include seocalcitol (EB1089 or CB1093),lexacalcitol (KH1060), fenretinide, Panretin™ (alitretinoin), tretinoinincluding Atragen™ (liposomal tretinoin), Targretin™ (bexarotene),LGD-1550 and the like.

PARP inhibitors include ABT-888, olaparib, KU-59436, AZD-2281,AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231 and the like.

Plant alkaloids include vincristine, vinblastine, vindesine, vinorelbineand the like.

Proteasome inhibitors include Velcade™ (bortezomib), MG132, NPI-0052,PR-171 and the like.

Examples of immunologicals include interferons and otherimmune-enhancing agents. Interferons include interferon alpha,interferon alpha-2a, interferon alpha-2b, interferon beta, interferongamma-1a, Actimmune™ (interferon gamma-1b), interferon gamma-n1,combinations thereof and the like. Other agents include Alfaferone(IFN-α), BAM-002 (oxidized glutathione), Beromun™ (tasonermin), Bexxar™(tositumomab), Campath™ (alemtuzumab), CTLA4 (cytotoxic lymphocyteantigen 4), dacarbazine, denileukin, epratuzumab, Granocyte™(lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010(anti-CTLA-4), melanoma vaccine, mitumomab, molgramostim, Mylotarg™(gemtuzumab ozogamicin), Neupogen™ (filgrastim), OncoVAC-CL, Ovarex™(oregovomab), pemtumomab (Y-muHMFG1), Provenge™ (sipuleucel-T),sargaramostim, sizofiran, teceleukin, Theracys™ (BCG or BacillusCalmette-Guerin), ubenimex, Virulizin™ (immunotherapeutic, LorusPharmaceuticals), Z-100 (Specific Substance of Maruyama or SSM), WF-10(tetrachlorodecaoxide or TCDO), Proleukin™ (aldesleukin), Zadaxin™(thymalfasin), Zenapax™ (daclizumab), Zevalin™ (90Y-ibritumomabtiuxetan) and the like.

Biological response modifiers are agents that modify defense mechanismsof living organisms or biological responses, such as survival, growth ordifferentiation of tissue cells to direct them to have anti-tumoractivity, and include krestin, lentinan, sizofiran, picibanil,PF-3512676 (CpG-8954), ubenimex and the like.

Pyrimidine analogs include cytarabine (cytosine arabinoside, ara C orarabinoside C), doxifluridine, Fludara™ (fludarabine), 5-FU(5-fluorouracil), floxuridine, Gemzar™ (gemcitabine), Tomudex™(raltitrexed), triacetyluridine, Troxatyl™ (troxacitabine) and the like.

Purine analogs include Lanvis™ (thioguanine), Purinethol™(mercaptopurine) and the like.

Antimitotic agents include batabulin, epothilone D (KOS-862),N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,ixabepilone (BMS-247550), paclitaxel, Taxotere™ (docetaxel), larotaxel(PNU-100940, RPR-109881 or XRP-9881), patupilone, vinflunine, ZK-EPO(synthetic epothilone) and the like.

Ubiquitin ligase inhibitors include MDM2 inhibitors such as nutlins,NEDD8 inhibitors such as MLN4924, and the like.

A composition comprising crystallineN-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II (or prepared usingas API) can also be used as radiosensitizers that enhance the efficacyof radiotherapy. Examples of radiotherapy include, but are not limitedto, external beam radiotherapy (XBRT), teletherapy, brachytherapy,sealed-source radiotherapy, unsealed-source radiotherapy and the like.

Additionally or alternatively, a composition comprising crystallineN-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II (or prepared usingas API) can be administered in combination therapy with one or moreantitumor or chemotherapeutic agents selected from Abraxane™ (ABI-007),ABT-100 (farnesyl transferase inhibitor), Advexin™ (Ad5CMV-p53 vaccineor contusugene ladenovec), Altocor™ or Mevacor™ (lovastatin), Ampligen™(poly(I)-poly(C12U), a synthetic RNA), Aptosyn™ (exisulind), Aredia™(pamidronic acid), arglabin, L-asparaginase, atamestane(1-methyl-3,17-dione-androsta-1,4-diene), Avage™ (tazarotene), AVE-8062(combretastatin derivative), BEC2 (mitumomab), cachectin or cachexin(tumor necrosis factor), Canvaxin™ (melanoma vaccine), CeaVac™ (cancervaccine), Celeuk™ (celmoleukin), histamine including Ceplene™ (histaminedihydrochloride), Cervarix™ (AS04 adjuvant-adsorbed human papillomavirus (HPV) vaccine), CHOP (Cytoxan™ (cyclophosphamide)+Adriamycin™(doxorubicin)+Oncovin™ (vincristine)+prednisone), combretastatin A4P,Cypat™ (cyproterone), DAB(389)EGF (catalytic and translocation domainsof diphtheria toxin fused via a His-Ala linker to human epidermal growthfactor), dacarbazine, dactinomycin, Dimericine™ (T4N5 liposome lotion),5,6-dimethylxanthenone-4-acetic acid (DMXAA), discodermolide, DX-8951f(exatecan mesylate), eniluracil (ethynyluracil), squalamine includingEvizon™ (squalamine lactate), enzastaurin, EPO-906 (epothilone B),Gardasil™ (quadrivalent human papilloma virus (Types 6, 11, 16, 18)recombinant vaccine), Gastrimmune™, Genasense™ (oblimersen), GMK(ganglioside conjugate vaccine), GVAX™ (prostate cancer vaccine),halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101,IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonasexotoxin, interferon-α, interferon-γ, Junovan™ and Mepact™(mifamurtide), lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine(hexadecylphosphocholine), Neovastat™ (AE-941), Neutrexin™ (trimetrexateglucuronate), Nipent™ (pentostatin), Onconase™ (ranpirnase, aribonuclease enzyme), Oncophage™ (vitespen, melanoma vaccine treatment),OncoVAX™ (IL-2 vaccine), Orathecin™ (rubitecan), Osidem™ (antibody-basedcell drug), Ovarex™ MAb (murine monoclonal antibody), paclitaxelalbumin-stabilized nanoparticle, paclitaxel, Pandimex™ (aglyconesaponins from ginseng comprising 20(S)-protopanaxadiol (aPPD) and20(S)-protopanaxatriol (aPPT)), panitumumab, Panvac™-VF (investigationalcancer vaccine), pegaspargase, peginterferon alfa (PEG interferon A),phenoxodiol, procarbazine, rebimastat, Removab™ (catumaxomab), Revlimid™(lenalidomide), RSR13 (efaproxiral), Somatuline™ LA (lanreotide),Soriatane™ (acitretin), staurosporine (Streptomyces staurospores),talabostat (PT100), Targretin™ (bexarotene), Taxoprexin™(docosahexaenoic acid (DHA)+paclitaxel), Telcyta™ (canfosfamide,TLK-286), Temodar™ (temozolomide), tesmilifene, tetrandrine,thalidomide, Theratope™ (STn-KLH vaccine), Thymitaq™ (nolatrexeddihydrochloride), TNFerade™ (adenovector: DNA carrier containing thegene for tumor necrosis factor-α), Tracleer™ or Zavesca™ (bosentan),TransMID-107R™ (KSB-311, diphtheria toxins), tretinoin (retin-A),Trisenox™ (arsenic trioxide), Ukrain™ (derivative of alkaloids from thegreater celandine plant), Virulizin™, Vitaxin™ (anti-αvβ3 antibody),Xcytrin™ (motexafin gadolinium), Xinlay™ (atrasentan), Xyotax™(paclitaxel poliglumex), Yondelis™ (trabectedin), ZD-6126(N-acetylcolchinol-O-phosphate), Zinecard™ (dexrazoxane), zoledronicacid, zorubicin and the like.

In one embodiment, a composition comprising crystallineN-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II (or prepared usingas API), is administered in a therapeutically effective amount to asubject in need thereof to treat cancer.

Examples include, but are not limited to, acoustic neuroma, acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemia(monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma,myelomonocytic and promyelocytic), acute t-cell leukemia, basal cellcarcinoma, bile duct carcinoma, bladder cancer, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma,chordoma, choriocarcinoma, chronic leukemia, chronic lymphocyticleukemia, chronic myelocytic (granulocytic) leukemia, chronicmyleogeneous leukemia, colon cancer, colorectal cancer,craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma,dysproliferative changes (dysplasias and metaplasias), embryonalcarcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelialcarcinoma, erythroleukemia, esophageal cancer, estrogen-receptorpositive breast cancer, essential thrombocythemia, Ewing's tumor,fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma,heavy chain disease, hemangioblastoma, hepatoma, hepatocellular cancer,hormone insensitive prostate cancer, leiomyosarcoma, liposarcoma, lungcancer, lymphagioendotheliosarcoma, lymphangiosarcoma, lymphoblasticleukemia, lymphoma (Hodgkin's and non-Hodgkin's), malignancies andhyperproliferative disorders of the bladder, breast, colon, lung,ovaries, pancreas, prostate, skin and uterus, lymphoid malignancies ofT-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma,medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma,myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, non-smallcell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma,ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillarycarcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer,renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma,sebaceous gland carcinoma, seminoma, skin cancer, small cell lungcarcinoma, solid tumors (carcinomas and sarcomas), small cell lungcancer, stomach cancer, squamous cell carcinoma, synovioma, sweat glandcarcinoma, thyroid cancer, Waldenström's macroglobulinemia, testiculartumors, uterine cancer and Wilms' tumor in a mammal,

In a more particular embodiment, a composition comprising crystallineN-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II (or prepared usingas API), is administered in a therapeutically effective amount to asubject in need thereof to treat myelodysplastic syndrome, acute myeloidleukemia, colorectal cancer, non-small cell lung cancer, and ovariancancer.

In still further embodiments of the invention, there is provided amethod for treating cancer in a mammal comprising dissolvingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II in apharmaceutically acceptable solvent or mixture of solvents, andadministering the resulting solution in a therapeutically effectiveamount to subject having the disease.

In still further embodiments of the invention, there is provided amethod for treating cancer in a mammal comprising dispersingN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I or hydrogen citrate Form II in apharmaceutically acceptable polymeric carrier, and administering theresulting solid dispersion in a therapeutically effective amount tosubject having the disease.

EXAMPLES

The following examples are merely illustrative, and do not limit thisdisclosure in any way.

PXRD data were collected using a G3000 diffractometer (Inel Corp.,Artenay, France) equipped with a curved position sensitive detector andparallel beam optics. The diffractometer was operated with a copperanode tube (1.5 kW fine focus) at 40 kV and 30 mA. An incident beamgermanium monochrometer provided monochromatic radiation. Thediffractometer was calibrated using the attenuated direct beam atone-degree intervals. Calibration was checked using a silicon powderline position reference standard (NIST 640c). The instrument wascomputer controlled using the Symphonix software (Inel Corp., Artenay,France) and the data was analyzed using the Jade software (version 6.5,Materials Data, Inc., Livermore, Calif.). The sample was loaded onto analuminum sample holder and leveled with a glass slide.

Example 1 Preparation ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I

N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureafree base solids (399.87 mg) was added to 20 mL of acetone:water mixture(75:25 v:v) and heated with stirring at 70° C. for 30 minutes. Thesample remained as a suspension. Citric acid (1.2 mL, 1 M) solution inwater was added with continued stirring at 70° C. At the end ofapproximately one hour, the sample was almost completely dissolved. Thesample was allowed to cool slowly to ambient temperatures overnight. Thesuspension was filtered through a 0.22 micron PTFE syringe filter. Thefiltrate was evaporative cooled by applying vacuum. Amorphous materialprecipitated forming a milky suspension, which turned into a “gummy”material (sticky mass) after several minutes. The sample was sonicatedfor approximately thirty minutes, upon which crystallization occurred.The crystalline solid was collected by vacuum filtration.

Example 2 Preparation ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureadihydrogen citrate Form I

N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureafree base solids (398.06 mg) was added to 20 mL of acetone:water mixture(75:25 v:v) and heated with stirring at 70° C. for 30 minutes. Thesample remained as a suspension. Citric acid (1.2 mL, 1 M) solution inwater was added with continued stirring at 70° C. After adding thecitric acid solution, slight more of the solid was observed to dissolve.Within 12 minutes after the addition of citric acid, a suspension wasformed. The suspension was stirred at 70° C. for approximately one hour.The solid was isolated by vacuum filtration.

TABLE 1 PXRD Peak Listing: N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea dihydrogen citrate Form I Peak Position (°2 θ)7.560 7.959 11.797 12.421 13.435 14.587 15.657 15.953 16.686 21.38122.396 22.815 23.983 24.504 24.993 26.122

Example 3 Preparation ofN-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureahydrogen citrate Form II

N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)ureafree base solid (102.65 mg) was dissolved in 5 mL of THF/water mixture(80/20 v/v) at 70° C. Citric acid (21.94 mg) was added to the solutionfollowed by the slow addition of heptane (7.5 mL). The appearance of asmall amount of precipitant formed in the heptane layer was noted. Theprecipitant dissolved as it fell into the THF/water layer. The solutionwas cooled to ambient temperatures. Crystalline solids formed afterseveral days at ambient temperatures. Solid was collected by vacuumfiltration.

TABLE 2 PXRD Peak Listing: N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea hydrogen citrate Form II Peak Position (°2 θ)5.71 6.30 7.27 8.65 9.80 13.06 14.31 15.18 15.92 16.68 17.71 20.17 21.9823.30 24.47 26.11 28.08

What is claimed is:
 1. The compound N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea dihydrogen citrate in a solid crystalline form, wherein the crystalline form is Form I, characterized at least by a powder X-ray diffraction peak at any one or more of the following positions: 11.80, 14.59, 15.95, 21.38, 26.12° 2θ, ±0.2° 2θ.
 2. The compound of claim 1, wherein the crystalline form is Form I, characterized at least by a powder X-ray diffraction peak at each of said positions.
 3. The compound of claim 1, wherein the crystalline form is Form I, characterized at least by a powder X-ray diffraction peak at each of said positions: 7.56, 7.96, 11.80, 12.42, 13.44, 14.59, 15.66, 15.95, 16.69, 21.38, 22.40, 22.82, 23.98, 24.50, 24.99, 26.12° 2θ, ±0.2° 2θ.
 4. The compound of claim 1, wherein the crystalline form is a monohydrate.
 5. A pharmaceutical composition comprising the compound of claim 1 and one or more pharmaceutically acceptable excipients.
 6. A process for preparing N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea dihydrogen citrate in a solid crystalline form, wherein the crystalline form is Form I of claim 1, comprising: a) providing a mixture comprising (i) N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea, water, acetone, and citric acid; b) causing N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea dihydrogen citrate crystalline Form I to exist in the mixture.
 7. The process of claim 6, further comprising isolating N-(4-{4-amino-7-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl}phenyl)-N′-(3-fluorophenyl)urea dihydrogen citrate in a solid crystalline form, wherein the crystalline form is Form I. 